Bacteria have evolved complex strategies to compete and communicate in their environments. A new mechanism of inter-bacterial competition, termed contact- dependent growth inhibition (CDI) was recently discovered in Escherichia coli. CDI systems are found in a wide variety of gram-negative bacteria, including several important human pathogens. CDI is mediated by the CdiB/CdiA two-partner secretion system. CdiB is a predicted outer membrane protein that is required for the export and assembly of the CdiA exoprotein onto the cell surface. The C-terminal domain of CdiA (CdiA-CT) contains the growth inhibition activity and is presumably cleaved and translocated into the target cell cytoplasm to inhibit growth. CDI systems also encode CdiI immunity proteins, which bind and inactivate CdiA-CT toxins, thereby protecting CDI+ cells from autoinhibition. Remarkably, the CdiA-CT domain is polymorphic, with well over 60 different toxin sequences identified to date. Accordingly, the corresponding CdiI proteins are also highly variable. This sequence diversity suggests that CDI systems deploy a wide variety of toxic activities. Indeed, we have discovered that CdiA- CT domains exhibit a number of distinct nuclease activities. Because CdiI immunity proteins are specific for their cognate CdiA-CTs, the interactions underlying each toxin- immunity complex are presumably unique. There is currently no structural information available for any CdiA-CT/CdiI complex, and therefore the details of these protein- protein interactions are completely unknown. Moreover, the mechanisms by which CdiI proteins neutralize CdiA-CT activities are not understood. We propose structural and functional analyses to gain insights into the intricate toxin-immunity network encoded by bacterial CDI systems, which represents a unique opportunity to elucidate how specific binding is maintained as toxin-immunity pairs diverge through evolution. PUBLIC HEALTH RELEVANCE: Bacteria have evolved complex strategies to compete and communicate in their environments. A new mechanism of inter-bacterial competition, termed contact-dependent growth inhibition (CDI) has recently been discovered in a wide variety of gram-negative bacterial pathogens. This proposal utilizes a structure/function analysis to gain mechanistic insights into the intricate toxin-immunity protein network encoded by CDI systems. This research will have a significant impact on our understanding of bacterial pathogenesis and ecology and could lead to the development of novel antimicrobial therapies.